Haploid lines of cultured cells from Rana pipiens will be employed in an attempt to resolve the relative roles of gene mutation and non- mutational clonal inheritance in the origin of cell culture "variants". This information is needed to design efficient procedures for the induction and isolation of haploid mutants and thus to realize the full potential for genetic studies of the haploid frog cell strains. We propose to isolate in sequence variants that show resistance to successively higher concentrations of purine and pyrimidine analogs. At each step, we will determine ploidy sensitivity, i.e., the dependence of the yield of variant colonies on the number of chromosome sets per cell. Defects in purine and pyrimidine salvage pathways behave as recessives in other cell culture systems, so that we anticipate that true mutants will show a large difference in haploid: diploid comparisons. Variants obtained at each selective step will be characterized by studies of karyotype, of transport kinetics, and in respect to properties of the target phosphorylating enzymes: activity, intracellular localization, thermal stability, electrophoretic mobility. Alterations that appear to be mutational by these criteria will be used in the development of an optimized protocol for mutagenesis, which will in turn be used to induce other classes of mutants, e.g., auxotrophs and temperature-sensitives. The strains resistant to base analogs will also be employed to determine appropriate conditions for the fusion of the cells, so that it will be possible to screen for complementation groups among classes of phenotypically similar variants. Chromosome maps will be prepared by banding techniques, to determine whether chromosome aberrations play a role in the origin of non-mutational variants.